We have shown previously that estrogen-stimulated transcription from the human lactoferrin gene in RL95-2 endometrium carcinoma cells is mediated through an imperfect estrogen response element (ERE) at the 5-flanking region of the gene. Upstream from the ERE, a DNA sequence (؊418 to ؊378, FP1) was selectively protected from DNase I digestion by nuclear extracts from endometrial and mammary gland cell lines. In this report, using the electrophoresis mobility shift assay, site-directed mutagenesis, and DNA methylation interference analyses, we show that three different nuclear proteins bind to the FP1 region (C1, C2, and C3 sites). The nuclear receptor, COUP-TF, binds to the C2 site. Mutations in the C1 binding region abolish C1 complex formation and reduce estrogen-dependent transcription from the lactoferrin ERE. When the imperfect ERE of the lactoferrin gene is converted to a perfect palindromic structure, the enhancing effect of the C1 binding element for estrogen responsiveness was abolished. We isolated a complementary DNA (cDNA) clone from an RL95-2 expression library that encodes the C1 site-binding protein. The encoded polypeptide maintains 99% amino acid identity with the previously described orphan nuclear receptor hERR1. A 2.2-kilobase mRNA was detected in RL95-2 cells by the newly isolated cDNA but not by the first 180 base pair of the published hERR1 sequence. By Western analysis, a major 42-kDa protein is detected in the RL95-2 nuclear extract with antibody generated against GST-hERR1 fusion protein. Finally, we show that the hERR1 interacts with the human estrogen receptor through protein-protein contacts.

BackgroundFood frequency questionnaire (FFQ) is a reliable tool to estimate dietary intake in large nutritional epidemiological studies, but there is lack of a current and validated FFQ for use in urban Chinese pregnant women. This study aimed to evaluate the reproducibility and validity of a semi-quantitative FFQ designed to estimate dietary intake among urban pregnant women in a cohort study conducted in central China.MethodsIn the reproducibility study, a sample of 123 healthy pregnant women completed the first FFQ at 12–13 weeks gestation and the second FFQ 3–4 weeks later. To validate the FFQ, the pregnant women completed three 24-h recalls (24HRs) between the intervals of two FFQs.ResultsThe intraclass correlation coefficients of two administrations of FFQ for foods ranged from 0.23 (nuts) to 0.49 (fruits) and for nutrients from 0.24 (iodine) to 0.58 (selenium) and coefficients were all statistically significant. The unadjusted Pearson correlation coefficients between two methods ranged from 0.28 (beans) to 0.53 (fruits) for foods and from 0.15 (iodine) to 0.59 (protein) for nutrients. Energy-adjusted and de-attenuated correlation coefficients for foods ranged from 0.35 (beans) to 0.56 (fruits) and for nutrients from 0.11 (iodine) to 0.63 (protein), and all correlations being statistically significant except for iodine, sodium and riboflavin. On average, 67.0 % (51.2 %-80.5 %) of women were classified by both methods into the same or adjacent quintiles based on their food intakes, while 68.5 % (56.1 %-77.2 %) of women were classified as such based on nutrient intakes. Extreme misclassifications were very low for both foods (average of 2.0 %) and nutrients (average of 2.2 %). Bland-Altman Plots also showed reasonably acceptable agreement between two methods.ConclusionThis FFQ is a reasonably reliable and valid tool for assessing most food and nutrient intakes of urban pregnant women in central China.

Catecholamines may influence vascular smooth muscle cell (SMC) growth and vascular hypertrophic diseases. We previously demonstrated that stimulation of alpha1-adrenoceptors (AR) causes hypertrophy of vascular SMCs in vitro and in situ. Here, we used adult rat aorta SMCs that express alpha1D- and alpha1B-ARs (but not alpha1A-ARs) in vitro to examine the mechanisms and alpha1-AR subtypes involved. Norepinephrine (NE) increased protein synthesis and content in a time- and dose-dependent manner. To identify the responsible alpha1-AR subtype, we first documented the selectivity of two alpha1-AR subtype antagonists, BMY 7378 (alpha1D-AR antagonist) and chloroethylclonidine (CEC; alpha1B-AR antagonist), using Rat-1 fibroblasts stably transfected with the three different rodent alpha1-AR cDNAs. NE dose-dependently increased protein synthesis in each cell line. In alpha1D fibroblasts, BMY 7378 inhibited growth and protected alpha1D-ARs from CEC alkylation while having little blocking or protecting effect on the growth induced by stimulation of fibroblasts that express alpha1A- or alpha1B-ARs. In rat aorta SMCs, pretreatment with CEC in the presence of BMY 7378 to protect alpha1D-ARs had no effect on NE-induced protein synthesis. BMY 7378 inhibited the SMC growth response with a pKb of 8.4. NE caused rapid and transient p42-p44 mitogen-activated protein kinase (MAPK) activation that was alpha1D-AR dependent. Furthermore, NE caused tyrosine phosphorylation of multiple cellular proteins, phosphorylation of Raf-1, and stimulation of c-fos mRNA expression in aorta SMCs. The selective MAPK kinase inhibitor PD 98059 inhibited NE-induced protein synthesis and MAPK activation with IC50 values of 2.3 and 1.6 microM, respectively. These data demonstrate that SMC growth induced by NE is mediated by alpha1D-ARs that couple to activation of the MAPK cascade.

Evidence suggests that the sympathetic nervous system and ␣ 1 -adrenoreceptors (AR) 1 may exert trophic influences over SMCs during normal development and also contribute to the pathogenesis of vascular hypertrophy and atherosclerosis (1, 2). Hyperinnervation of blood vessels by catecholaminergic fibers in the genetic spontaneously hypertensive rat has been correlated with SMC hypertrophy and hyperplasia in these vessels (3). Also, sympathectomy attenuates normal growth, as well as hypertrophy of the vascular wall in hypertensive animals (4 -7). There is considerable evidence that smoking, stress, and hypertension, which are key risk factors for atherosclerosis and hypertrophic vascular disease, are associated with elevated plasma catecholamines (8, 9).Catecholamines have been shown to initiate not only immediate SMC responses such as contraction of blood vessels, but may also influence proliferation and growth of cultured vascular SMCs (10 -13). In nonconfluent, cultured rat and rabbit aortic SMCs, AR stimulation promotes cell proliferation (10, 13). Furthermore, ␣ 1 blockade reduces vascular collagen synthesis in the spontaneous hypertensive rat (14), and inhibits SMC proliferation induced by endothelial denudation (13, 15) and angiotensin infusion (16) in normal rats. In cholesterol-fed monkeys, elevated plasma norepinephrine (NE) greatly increased atherosclerotic lesion growth (17). Infusion of NE over a 2-week period, at a level which did not cause sustained elevation of blood pressure, induced formation of atherosclerotic vascular lesions in rabbit aorta (18). There is also evidence that ␣ 1 ARs mediate growth of myocardial cells. In cultured neonatal rat cardiac myocytes that possess both ␤ 1 and ␣ 1 ARs, stimulation of ␣ 1 ARs with NE increased cell protein, RNA, myocyte surface area, and contractile protein expression (19). However, no studies have examined whether ␣ 1 ARs influence proliferation-independent growth of SMCs.Both molecular cloning and pharmacologic studies have shown that ␣ 1 ARs are comprised of three closely related subtypes (20, 21). We have recently used polymerase chain reaction (22) and RNase protection assays 2 to determine expression of ␣AR subtype mRNA by rat vascular SMCs. Freshly isolated and early passage cultured aortic and vena cava SMCs express both ␣ 1B and ␣ 1D mRNA (20), and it appears that both receptors are present on SMCs of rat aorta (24,25, and Ref. 22 and references therein). Rat aorta has been shown recently to also express ␣ 1A (formerly denoted "␣ 1C ") mRNA (26 -29). Given this multiplicity of ␣ 1 AR expression, the purpose of the present study was first to examine both in vitro and in situ, the effects of combined stimulation of the ␣ 1 AR subtypes with NE alone on proliferation-independent growth, and expression of sarcomeric ␣-SMC-actin and cytoskeletal ␤-actin mRNAs by arterial and venous SMCs. Second, effects of combined stimulation were compared with those during treatment with NE plus antagonists, 5-methylurapidil (5-MU, selectivity ϭ ␣ 1A Ͼ ␣ 1D Ͼ ␣ 1B...